Difference between revisions of "Team:Heidelberg/Cytochrome Engineering"

Cytochromes are heme-dependent enzymes of immense importance across all kingdoms of life. Due to their often highly promiscuous nature, the cytochrome P450 superfamily is of particular interest in context of enzyme engineering. In this subproject, we aimed at applying our PREDCEL toolbox for re-directing the catalytic activity of cytochromes towards desired products. Employing the caffeine-metabolizing human Cytochrome P450 1A2 (CYP1A2) as example, our team implemented a PREDCEL enzyme evolution workflow coupling phage survival to the production of a naturally unfavored catalytic product: theophylline. We created M13 phages encoding CYP1A2 as well as a corresponding accessory plasmid linking the intracellular theophylline levels to geneIII production via a theophylline riboswitch. After iterative propagation of the CYP1A2-encoding phages on mutagenic selection cells transformed with our accessory plasmid, we observed numerous, partially recurrent point mutations in CYP1A2, indicative of a successful evolution. Taken together, our work lays the foundation for the future engineering of enzymes by means of in vivo directed evolution with PREDCEL.